There are a number of human antiviral vaccines that are currently in use. These include Hepatitis A virus, Hepatitis B virus, Influenza virus, Japanese B encephalitis virus, Measles, Mumps, Rubella (MMR) virus, Poliovirus virus, Rabies virus, Smallpox, Varicella-Zoster, Yellow fever virus vaccines. In addition to the growing number of vaccine products, there are different compositions or formulations in use or being developed for a given vaccine. The successful use of live viral vaccines depends not only on the proper choice and delivery of the virus, but also on maintaining the sufficient titer or potency required for an immune response. The inherent lability of live viruses presents a particular formulation challenge in terms of stabilizing and preserving vaccine viability during manufacturing, storage, and administration. There are a number of formulations known in the art for rotavirus vaccines but suffer from one or the other problems with regard to stability during storage.
Rotavirus is a genus of double-stranded RNA viruses in the family Reoviridae and is transmitted by the faecal-oral route. It infects cells that line the small intestine and produces an enterotoxin, which induces gastroenteritis, leading to severe diarrhea and sometimes death through dehydration. Rotavirus infection is the greatest cause of diarrhea-related deaths among infants and young children. Every year, rotavirus gastroenteritis causes the death of 310,000-590,000 infants and young children worldwide.
All rotavirus vaccines developed to date have been based on live rotavirus strains that have been isolated from humans or animals and in vitro reassorted, adapted to cell cultures, and then formulated for oral delivery. Both monovalent and multivalent animal-based strains have demonstrated efficacy as candidate vaccines.
The human rotavirus strain 116E, a natural human-bovine reassortant and naturally attenuated, is a human G9 strain into which a single bovine VP4 gene (VP=viral protein), homologous to the P[11] gene segment, was naturally introduced. The I132 strain, also named G10P [11], is primarily composed of bovine genes and has only two gene segments of human origin, VP5 and VP7. These two rotavirus vaccine strains have been individually prepared as pilot lots of monovalent oral rotavirus vaccine liquid formulations for clinical trials to be conducted in India.
Bharat Biotech International Ltd. (BBIL) obtained the human rotavirus strains, 116E and I321 from National Institute of Health (NIH) under the material transfer agreement with National Institute of Allergy and Infectious Diseases (NIAID), NIH, Bethesda, USA. The original 116E (G9[P11]) and I321 (G10P[11]) were adapted to grow in cell culture by passages in primary African green monkey kidney (AGMK) cells then in MA104 cell substrate and later in serially Passaged AGMK (SPAGMK). MA104 and SPAGMK cell substrates are not approved by National Regulatory Authorities (NRA) for commercial vaccine production. Hence it is preferable to adapt 116E and I321 and other rotavirus vaccine strains to approved, certified, licensed and fully characterized cell substrate like Vero cell substrate and/or human diploid cells like MRC-5.
The prior art known to the applicant includes WO 02/11540 A1 describes rotavirus vaccine formulations, which include buffering agents appropriate for oral administration of rotavirus vaccines. The formulations disclosed in WO 02/11540 A1 also include compounds to stabilize the vaccine compositions against potency loss. More specifically, the compositions disclosed in WO 02/11540 require a sugar, phosphate and at least one carboxylate at least one human serum albumin or amino acid selected from glutamate, glutamin and arginin. However, the stabilities achieved varied greatly, especially at temperatures over 20° C. appear to show considerable losses in potency with the formulations of WO 02/11540 A1.
WO 99/62500 ('500), WO 2005/058356 ('356) A 2 and WO 2001/012797 ('197) discloses using vaccine stabilizers for preparing vaccine formulations and lyophilized vaccines, storage stable virus compositions, method of separating rotavirus variants and live attenuated rotavirus Liquid Vaccine. '500 discloses measles-mumps-rubella lyophilised vaccine prepared employing stabilizer consisting of hydrolysed gelatin, sorbitol, phosphate, sodium chloride, sucrose, bicarbonate, glucose human serum albumin and citrate. The invention banks upon dual presence of increased amount of a disaccharide and polyhydric alcohol at pH between 6.0 and 7.0 for thermo-stability.
Despite requiring number of ingredients making the invention cost extensive, fails to achieve stability at ambient temperatures. This in turn adds to the requirement of special infrastructure for storing the vaccine making the invention further costly. However, most of these formulations offer limited storage stability and thus are not commercially viable.
PCT/IN07/00190 titled A Composition Useful as a Vaccine discloses a stable vaccine The essence of the invention centers around the combined effect of the first protein that is human serum albumin, the second protein, which is at least partially hydrolyzed and a combination of three different sugars. Additionally, the inventions also rely on inclusion of trypsin in the culture medium during adaptation of viruses. The claimed vaccine is stable for 3 weeks at 37° C., six months at 25° C., and one year at 2° C.-8° C.
It is apparent from the foregoing description that despite these advances in area of vaccine formulation, there remains a distinct need for live commercially viable viral vaccine with improved thermo-stability and shelf life.
The present invention fulfills this need by providing live or live attenuated virus that exhibits better and improved stability characteristics whether in the form of a pooled bulk from three single harvests of the virus from the same batch, or in a liquid or lyophilized formulation. Stability with reference to the virus (e.g., rotavirus or rotavirus vaccine) herein shall mean viral titer at a given point in time starting from the time of harvest from the cultured cells through the bulk stage to the formulated vaccine. The inventors after prolonged research could develop a composition of the present invention useful as a vaccine that exhibit enhanced stability of the bulk and formulated particularly at ambient temperature.
An improved stability, in statistically significant terms, can be achieved by the use of the virus that has come in contact with or been exposed to human serum albumin during the virus growth and multiplication stage in cell cultures. For purposes of this invention, the virus is deemed to come in contact with or be exposed to human serum albumin when the virus infected host cells are propagated in a cell culture medium/growth medium supplemented with human serum albumin. The virus or virus population that has been so exposed to human serum albumin is referred to as a “pre-conditioned” virus. The virus that has not been so exposed to the human serum albumin is referred to as a “typical virus” herein. The pre-conditioned virus whether at the bulk stage or in the form of a formulation, i.e., a vaccine/formulated vaccine, exhibits better stability (in statistically significant terms) than the typical virus.
The present invention further discloses that the stability of the virus, whether it is the pre-conditioned virus or the typical virus, in a formulation can also be further improved or at least sustained i.e., the stability can be maintained or, at a minimum, delayed from gradually reaching nil or zero stability during storage by practicing systems (i) and (ii): According to system (i), to realize improved or sustained stability, the virus is formulated with a non-viral protein or protein hydrolysate thereof or a vegetable protein or an analogous protein such as human serum albumin. The hydrolysate can be exemplified but not limited to lactalbumin hydrolysate, yeast hydrolysate, peptone, gelatin hydrolysate, and egg protein hydrolysate. The vegetable protein includes but not restricted to corn protein, wheat protein, garbanzo bean protein, kidney bean protein, lentil protein, lima bean protein, navy bean protein, soybean protein, split pea protein. Human serum albumin is of natural or recombinant origin. The virus is formulated with the non-viral protein or protein hydrolysate thereof simply by supplementing the formulation used for making the vaccine with the non-viral protein or protein hydrolysate thereof. This system (i) is understood to mean a single component system. According to system (ii), the virus is contacted with a non-viral protein or protein hydrolysate thereof as in the single component system, and 1-2 disaccharides by supplementing the formulation used for making the vaccine with protein or protein hydrolysate and 1-2 disaccharides. This system (ii) is understood to mean a two or three component system depending on whether the formulation containing virus is supplemented with a single disaccharide (two component system) or a combination of two different disaccharides (three component system). By practicing system (ii), the stability levels seen in the single component system is further improved.
Thus, in one general aspect, the present invention discloses compositions containing pre-conditioned virus or typical virus exhibiting improved and/or sustained stability.
The novelty of the invention resides in supplementing the culture medium with human serum albumin while propagating virus to achieve the viral antigen and vaccine formulation with enhanced titer value, shelf life, and thermostability even without supplementation of stabilizers. The shelf life can be further enhanced with addition of stabilizers as disclosed herein before. This leads to a therapeutically better vaccine adopting simple cost effective commercially viable process. In addition to technical advancement, the invention also qualifies the acid test of economic significance.